In recent years, an antibody-drug conjugate (Antibody-Drug Conjugate: ADC), in which a drug and an antibody are connected through a linker and which is capable of actively transporting the drug to an antigen-presenting cell, is practically used and has attracted high attention in the field of pharmaceuticals (Toxins, 2011, 3, p. 848-883 (Non-Patent Document 1), J. Med. Chem., 2011, 54, p. 3606-3623 (Non-Patent Document 2)).
One of the linker materials for ADC which have been utilized is a hetero type monodispersed polyethylene glycol. The hetero type monodispersed polyethylene glycol is a monodispersed polyethylene glycol, which contains as a main component, a hetero type polyethylene glycol having functional groups different from each other at both terminals thereof and has a definite molecular weight.
In the ADC, since an antibody and a drug are separately conjugated to each of the terminals of the hetero type polyethylene glycol as a linker, when a compound having the mutually same functional groups at the both terminals (homo type polyethylene glycol or the like) is present as an impurity in the hetero type monodispersed polyethylene glycol, a compound having two antibodies conjugated or a compound having two drugs conjugated is generated. The compound having two antibodies conjugated does not exhibit the effect of ADC because the drug is not conjugated. The compound having two drugs conjugated is transported to a position other than the antigen-presenting cell to be a cause of side effects because the antibody is not conjugated. Further, the same problems as described above also arise in the case where other hetero type compound having a different combination of functional groups from the desired combination of functional groups is present as an impurity, because a compound losing either the desired antibody or the desired drug is generated. Therefore, from the standpoint of use and effect of the drug, it can be said to be important that the hetero type monodispersed polyethylene glycol contains only one kind of hetero type polyethylene glycol having the different functional groups each other at the both terminals thereof in high purity.
Further, in recent years, for the purpose of increasing the effect of ADC, it has been attempted to use ADC in which a plurality of drugs are conjugated to an antibody. In the production of such ADC, the number of the drugs conjugated is ordinarily confirmed by using a mass spectrometer and HPLC. Therefore, when a compound having a different ethylene glycol chain length is present as an impurity in the hetero type monodispersed polyethylene glycol which is used as the linker material, a problem arises on the production in that the confirmation thereof becomes difficult. In addition, when the compound having a different ethylene glycol chain length is present as an impurity, there is a problem in that since the equivalents of the antibody and drug to be added in the production of ADC become uncertain, it is necessary to use excess amount of the expensive antibody and drug, and there is a problem in that since the compound having a different ethylene glycol chain length is treated as a different compound from the main agent at the time of drug application, identification of the compound, implementation of various tests, evaluation of permissible amount and the like are further required. Therefore, it can be said to be important that the hetero type monodispersed polyethylene glycol contains only one kind of polyethylene glycol having the same ethylene glycol chain length in high purity.
Thus, it has been desired for the hetero type monodispersed polyethylene glycol used as the linker material for ADC to contain as the main component, a compound of a hetero type polyethylene glycol having functional groups different from each other at both terminals thereof, in which the ethylene glycol chain length of the hetero type polyethylene glycol is the same, in particularly high purity.
Further, as the functional group, for example, a maleimide group or an iodoacetamide group, each of which reacts with a thiol group of antibody, drug or the like; a carboxyl group, an active ester group, an active carbonate group or an aldehyde group, each of which reacts with an amino group of antibody, drug or the like; an amino group, which reacts with a carboxyl group of antibody, drug or the like; or an azido group or an alkynyl group, each of which is used in a click reaction is used. Among them, since a hetero type polyethylene glycol having an amino group and a carboxyl group at each terminal is able to be used as it is, as the linker for ADC and is also able to be used as a raw material for functional group transformation, it is a useful compound as the hetero type monodispersed polyethylene glycol.
As the method for producing the hetero type monodispersed polyethylene glycol, a method containing a terminal functionalization step and an extension step of ethylene glycol chain length by Williamson ether syntheses is exemplified. For example, in U.S. Pat. No. 5,672,662 (Patent Document 1) or Japanese National Publication of International Patent Application No. 2007-538111 (Patent Document 2), a method of introducing a carboxyl group into a terminal of monomethoxypolyethylene glycol is disclosed. In Patent Document 1, monomethoxypolyethylene glycol and acrylonitrile are reacted by Michael addition reaction, the nitrile is converted to an amide under conditions of concentrated hydrochloric acid, and the amide is hydrolyzed under conditions of an aqueous potassium hydroxide solution, thereby introducing a carboxyl group. However, the purity and the yield are insufficient, because under the conditions of such strong acid and strong base, a compound having a hydroxyl group instead of a carboxyl group is generated by a reverse reaction of the Michael addition reaction or a compound having a short ethylene glycol chain length is generated by cleavage of the ethylene glycol chain. As to the purity, in the comparative example of Patent Document 2, the result of performing double check is described and it is confirmed to generate a compound without a methoxy group at the terminal. On the other hand, in Patent Document 2, monomethoxypolyethylene glycol and tert-butyl acrylate are reacted by Michael addition reaction and a carboxyl group is introduced under conditions of trifluoroacetic acid. However, in the technique described in the document, the introduction ratio of tert-butyl acrylate is low as 70% or less and a problem arises in that a compound having a hydroxyl group at the terminal remains.
Further, in the methods described in Patent Documents 1 and 2, monomethoxypolyethylene glycol is used as the raw material and in the case of using the reaction described above, a compound having a short ethylene glycol chain length and a compound having a hydroxyl group instead of a carboxyl group at the terminal are also generated as impurities in the case of obtaining a hetero type monodispersed polyethylene glycol. When the hetero type monodispersed polyethylene glycol containing such impurities is used in the production of ADC, the problem arises on the production of ADC described above based on the compound having a short ethylene glycol chain length and a compound losing either the antibody or the drug is generated due to the compound having a hydroxyl group at the terminal to cause decrease in effectiveness as drug.
Further, for example, the difference between the compound having a hydroxyl group at the terminal and the desired compound having a carboxyl group at the terminal resides only in the structure of the terminal so that it is difficult to perform separation and purification. In Patent Document 2, although the resulting product is purified using anion exchange chromatography, there is a problem from the standpoint of convenience and yield, because the method using anion exchange chromatography is a low versatility purification method and the yield decreases further.
Moreover, in the case of producing a hetero type monodispersed polyethylene glycol containing a hetero type polyethylene glycol having an ethylene glycol chain length of 5 or more, an extension reaction of the ethylene glycol chain length by Williamson ether synthesis is ordinarily necessary. This is because triethylene glycol or tetraethylene glycol, in which the ethylene glycol chain length is 4 or less, is able to be subjected to distillation purification so that the raw material having purity of 98% or more is available, but the compound having an ethylene glycol chain length of 5 or more cannot be subjected to distillation purification so that it is difficult to obtain a high purity product at a low cost. It is known that during the extension reaction of the ethylene glycol chain length by Williamson ether synthesis, by-products are generated by the side reaction. For example, as described in Tetrahedron, 1997, 53, p. 10939-10952 (Non-Patent Document 3), a compound having an ethylene glycol chain length less than the ethylene glycol chain length of the desired compound by 1 due to elimination of ethylene glycol is generated and compounds different in the ethylene glycol chain length and the terminal functional groups are generated by E2 elimination. In fact, in Angew. Chem. Int. Ed., 2009, 48, p. 1248-1252 (Non-Patent Document 4), it is described that a compound having an ethylene glycol chain length less than the ethylene glycol chain length of the desired compound by 1 due to elimination of ethylene glycol is generated in a yield of 3% at the time of Williamson ether synthesis. When a hetero type monodispersed polyethylene glycol is produced using a mixture containing such a compound having an ethylene glycol chain length less than the ethylene glycol chain length of the desired compound by 1, the hetero type monodispersed polyethylene glycol obtained contains the compound having an ethylene glycol chain length less than the ethylene glycol chain length of the desired main component by 1 as an impurity. Therefore, when such a hetero type monodispersed polyethylene glycol is used in the production of ADC, the problem arises on the production of ADC described above. The compounds having the ethylene glycol chain length different from each other are difficult to perform separation and purification because of similarity in the structure of the compounds, and as described in Non-Patent Document 4, it is necessary to perform multiple times of reverse phase chromatography or the like.
Further, in order to produce the hetero type polyethylene glycol having functional groups different from each other at the both terminals thereof, it is useful to use an intermediate for the production of a hetero type monodispersed polyethylene glycol having a protective group or a leaving group at one terminal and a hydroxyl group at the other terminal. As a method of obtaining such an intermediate, for example, in Org. Lett., 2002, 4, p. 2329-2332 (Non-Patent Document 5), a method in which tetraethylene glycol or hexaethylene glycol is used as a starting material and one terminal thereof is selectively tosylated using silver oxide is disclosed and it is described that a one terminal tosylated compound can be efficiently obtained by using silver oxide. However, this method has a problem in that a compound having tosyl groups at both terminals is generated in a yield of several percent.
Moreover, in Polym. Chem., 2014, 5, p. 694-697 (Non-Patent Document 6), synthesis of a one terminal Dmtr compound having an ethylene glycol chain length of 8 is disclosed. A part of the synthetic route is represented by following formula:DmtrO—(CH2CH2O)4-Ts+HO—(CH2CH2O)4—H→DmtrO—(CH2CH2O)8—H+DmtrO—(CH2CH2O)12-Dmtr(in the formula, Dmtr represents a 4,4′-dimethoxytrityl group.)
In Non-Patent Document 6, it is described that in the case of obtaining a one terminal Dmtr octamer by a 1:1 reaction of a one terminal tosyl compound with tetraethylene glycol, a both terminal Dmtr dodecamer by a 2:1 reaction of a one terminal tosyl compound with tetraethylene glycol is generated. However, when a hetero type polyethylene glycol having an amino group and a carboxyl group at both terminals respectively is synthesized using a mixture containing such a compound having a tosyl group and a Dmtr group at both terminals, a compound having amino groups at both terminals or a compound having carboxyl groups at both terminals is generated as an impurity. Therefore, when the hetero type monodispersed polyethylene glycol containing such an impurity is used in the production of ADC, a compound having two drugs conjugated or a compound having two antibodies conjugated is generated to cause decrease in effectiveness as drug.
On the other hand, as a method of evaluating the purity of the hetero type monodispersed polyethylene glycol, methods using 1H-NMR measurement, MS measurement, HPLC measurement and the like are exemplified. However, since a plurality of impurities are contained in the case of producing the hetero type monodispersed polyethylene glycol according to a known production method as described above, it can be said that it is difficult to evaluate the purity.
For example, in the case where a compound having amino groups at both terminals and a compound having an amino group and a hydroxyl group at both terminals respectively are intermixed in the hetero type monodispersed polyethylene glycol containing as a main component, a hetero type polyethylene glycol having an amino group and a carboxyl group at both terminals respectively, since an α-position proton of the hydroxyl group is overlap with the ethylene glycol chain and cannot be distinguished in 1H-NMR measurement, the content of the compound having an amino group and a hydroxyl group at both terminals respectively cannot be measured, and accordingly, it is also difficult to identify the content of the compound having amino groups at both terminals. Further, in MS measurement, although it is possible to identify a plurality of impurities, there is a problem of low quantitativity, because the efficiency of ionization varies depending on structure of the compound. Although it is possible to quantify by synthesizing a standard compound of the impurity and preparing a calibration curve, it is difficult that all of compounds including compounds having different ethylene glycol chain lengths and compounds having different combinations of the terminal functional groups are synthesized and quantified. Moreover, in HPLC measurement, it is possible to quantify if all impurities can be separated, but in the case where a plurality of compounds having different ethylene glycol chain lengths and compounds having different combinations of the terminal functional groups are present, the separation of these compounds is difficult.
Thus, it is difficult to evaluate the purity of the hetero type monodispersed polyethylene glycol and conventional evaluation methods have a problem in that as to the hetero type monodispersed polyethylene glycol, it is difficult to accurately measure the content of a compound in which the different combination of the functional groups at the both terminals from the desired compound and the content of a compound in which the different ethylene glycol chain length from the desired compound.